Functional Role of Pseudouridine on Zika Virus Gene Expression

假尿苷对寨卡病毒基因表达的功能作用

基本信息

批准号：
10732617
负责人：
Cara Theresia Pager
金额：
$ 22.8万
依托单位：
STATE UNIVERSITY OF NEW YORK AT ALBANY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-06-09 至 2025-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10732617
关键词：
Affect Affinity Americas Antisense Oligonucleotides Antiviral Agents Appearance Biological Assay Biotinylation Cell Line Cell Nucleus Cells Chemicals Culicidae Cytoplasm Defect Detection Development Disease Outbreaks Enzymes Flavivirus Gene Expression Genome Goals HIV-1 Health Hepatitis C virus Human Immune response Infection Knowledge Label Length Life Cycle Stages Luciferases Mass Spectrum Analysis Methodology Microcephaly Modification Molecular Molecular Biology Mutagenesis Neurologic Nuclear Export Nucleotides Plaque Assay Polyribosomes Positioning Attribute Proteins Pseudouridine RNA RNA Folding RNA Interference RNA Stability RNA Viruses RNA-Protein Interaction Regulation Replicon Reporting Research Reverse Transcription Role Site Site-Directed Mutagenesis Stress Structure System Techniques Technology Testing Therapeutic Time Transcript Translations Untranslated Regions Viral Viral Genes Viral Physiology Viral Proteins Virion Virus Virus Assembly Virus Diseases Western Blotting Woman ZIKA ZIKV infection Zika Virus combinatorial epitranscriptomics hepatoma cell improved infancy insight mosquito-borne new therapeutic target next generation sequencing novel overexpression therapeutic target transcriptome sequencing viral RNA viral transmission

项目摘要

Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus, that during the 2015-2016 outbreak in the Americas was associated with severe and devastating developmental abnormalities, including microcephaly, in babies born from infected women. The single-stranded positive-sense RNA genome of ZIKV directs translation of viral proteins, functions as a template for replication of the genome and is packaged into new virus particles. These functions are in part regulated by specific RNA structures within the untranslated regions of the RNA, and via interactions with viral and cellular proteins. More recently RNA modifications have been shown to significantly affect the virus infectious cycle. Although more than 170 different RNA modifications are known to decorate natural RNAs, only N6-methyladenosine has been reported to affect ZIKV gene expression. The role of other RNA modifications on ZIKV RNA is presently unknown. Using biotinylated antisense oligonucleotides to affinity isolate virus RNA from cells and virions, and mass spectrometry to identify RNA modifications, we discovered that more than 30 different RNA modifications are present on ZIKV RNA. In this application we focus on pseudouridine (Y), an abundant but understudied RNA modification. The incorporation of Y on cellular RNAs affects the folding, stability, and translation of RNA, and RNA-RNA and RNA-protein interactions. We propose that Y on the ZIKV RNA genome promotes virus translation and replication. In this application we propose using a next generation sequencing approach combined with chemical modification of pseudouridine to identify specific Y sites on ZIKV genome (Aim 1). Next, we will undertake RNAi depletion and overexpression of cellular pseudouridine synthase proteins to identify the host enzymes responsible for installing pseudouridine on ZIKV RNA (Aim 2). Last, we will use mutagenesis and modulation of the pseudouridine synthase enzymes to elucidate if pseudouridine regulates ZIKV translation and/or replication (Aim 3). Moreover, these studies will be undertaken in the context of virus infection in mammalian and mosquito cells, two hosts biologically relevant to the virus life cycle, which could inform the role of RNA modifications in virus transmission and host adaptation. We expect that this research will advance our knowledge of RNA modifications which have been shown to be novel and important regulators of viral gene expression and improve our understanding of the molecular biology of ZIKV. This research will therefore provide critical information on a virus with significant impacts on human health and that currently lacks therapeutic options.

Zika病毒（ZIKV）是一种重新出现的蚊子传播黄病毒，在2015 - 2016年爆发美洲爆发中，与受感染妇女出生的婴儿相关的严重而毁灭性的发育异常，包括小头畸形，包括小头畸形。 ZIKV的单链阳性RNA基因组指导病毒蛋白的翻译，作为复制基因组复制的模板，并被包装成新的病毒颗粒。这些功能部分受RNA未翻译区域内的特定RNA结构的调节，以及与病毒和细胞蛋白的相互作用。最近，RNA修饰已显示出显着影响病毒感染周期。尽管已知有170多种不同的RNA修饰可以装饰天然RNA，但据报道，仅N6-甲基读二糖苷会影响ZIKV基因表达。目前尚不清楚其他RNA修饰在ZIKV RNA上的作用。使用生物素化的反义寡核苷酸与细胞和病毒体的亲和力分离病毒RNA以及质谱法鉴定RNA修饰，我们发现在ZIKV RNA上存在30多种不同的RNA修饰。在此应用中，我们专注于伪苷（Y），这是一种丰富但研究了RNA的修饰。 Y在细胞RNA上的掺入会影响RNA以及RNA-RNA和RNA-蛋白质相互作用的折叠，稳定性和翻译。我们提出，ZIKV RNA基因组上的y促进了病毒的翻译和复制。在此应用中，我们建议使用下一代测序方法与假氨酸化学修饰相结合，以识别ZIKV基因组上的特定Y位点（AIM 1）。接下来，我们将进行细胞假氨酸合酶蛋白的RNAi耗竭和过表达，以鉴定负责在ZIKV RNA上安装假喹啉的宿主酶（AIM 2）。最后，我们将使用假氨酸合酶酶的诱变和调节，以阐明假氨酸是否调节ZIKV翻译和/或复制（AIM 3）。此外，这些研究将在哺乳动物和蚊子细胞的病毒感染中进行，这两个宿主与病毒生命周期具有生物学相关，这可以为RNA修饰在病毒传播和宿主适应中的作用提供信息。我们预计这项研究将提高我们对RNA修饰的了解，RNA修饰已被证明是病毒基因表达的新颖而重要的调节剂，并提高了我们对ZIKV分子生物学的理解。因此，这项研究将提供有关病毒的关键信息，对人类健康产生重大影响，目前缺乏治疗选择。